B. Protein Blotting

A general protocol for sample preparation.

Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.

Wash membrane again four times for 5 min each in TBST.

The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

Species Reactivity:

The leukocyte Ig-like receptor subfamily B (LILRB) are type-I transmembrane glycoproteins containing ligand binding extracellular IgG-like domains and immunoreceptor tyrosine-based inhibition motifs (ITIMS) within the cytoplasmic domain, which recruit SHP protein tyrosine phosphatases, leading to transduction of signals that inhibit immune cell activation. Encoded within a region of chromosome 19 known as the leukocyte receptor complex, the LILRB subfamily of inhibitory receptors consists of LILRB1 to LILRB5, also referred to as CD85J, CD85D, CD85A, CD85K, and CD85C, respectively (1). There is mounting evidence that LILRBs function, in part, as a novel class of immune checkpoint receptors and support tumor growth through the transmission of inhibitory signals upon engagement of ligands expressed on tumor cells (2).

LILRB1 (CD85j/ILT2/LIR1) is expressed across multiple hematopoietic cell lineages, such as B-cells, NK cells, monocytes, and T-cells (3,4). Like other members of the LILRB subfamily, LILRB1 contains multiple extracellular IgG-like domains and intracellular ITIM motifs (5). Research studies have demonstrated that LILRB1 interacts with multiple HLA class I ligands, such as HLA-G (6). Cross-linking of LILRB1 upon ligand engagement has been shown to activate immunosuppressive signaling cascades, which in B-cells, suppresses their activation and ability to produce antibodies (7). In NK and T cell lineages, research studies have shown that LILRB1 transduces signaling to inhibit cytolytic activity (3).